Adenoviral vector with shield and adapter increases tumor specificity and escapes liver and immune control

Abstract

Most systemic viral gene therapies have been limited by sequestration and degradation of virions, innate and adaptive immunity, and silencing of therapeutic genes within the target cells. Here we engineer a high-affinity protein coat, shielding the most commonly used vector in clinical gene therapy, human adenovirus type 5. Using electron microscopy and crystallography we demonstrate a massive coverage of the virion surface through the hexon-shielding scFv fragment, trimerized to exploit the hexon symmetry and gain avidity. The shield reduces virion clearance in the liver. When the shielded particles are equipped with adaptor proteins, the virions deliver their payload genes into human cancer cells expressing HER2 or EGFR. The combination of shield and adapter also increases viral gene delivery to xenografted tumors in vivo, reduces liver off-targeting and immune neutralization. Our study highlights the power of protein engineering for viral vectors overcoming the challenges of local and systemic viral gene therapies.

Innate immunity to adenovirus: lessons from mice

“…The most radical approach to avoiding HAdv recognition by the immune system has been through shielding the entire HAdv virion with PEG [24,[124][125][126][127], or coating the virus with engineered protein shields [128][129][130]. These approaches have demonstrated both a significantly improved persistence of HAdv in the circulation after intravenous virus administration and a reduction in the amount of inflammatory cytokines in the blood [48,131].…”

TRIM21—From Intracellular Immunity to Therapy

“…It should be noted however, that TRIM21 has also been implicated in both positive and negative regulation of innate signaling independent of antibody binding (112)(113)(114)(115)(116)(117). Furthermore, the identification of TRIM21 as a major player in antibody mediated block to gene therapy, together with the effect of complement factors C1 and C4, have inspired strategies to shield Ad5 gene delivery vectors from pre-existing immunity (19,110). The finding that such shielding strategies limit immune responses to the vector should be of particular interest in cases where repeated administrations are needed.…”

“…As hexon is the primary immunogen to which antibodies is generated, rh9C12 that binds to the apex of the hexon trimer spike (61), may be particularly well suited for such pre-coating. Indeed, shielding the Ad5 surface with a trivalent 9C12 scFv with high avidity has been used in combination with targeted designed ankyrin repeat proteins (DARPin's) binding to the fiber knob of an Ad5 vector (110). The DARPin units block CAR mediated uptake and re-targeted the vector to HER2 or EGFR positive tumor cells in mice (110,111).…”

“…Indeed, shielding the Ad5 surface with a trivalent 9C12 scFv with high avidity has been used in combination with targeted designed ankyrin repeat proteins (DARPin's) binding to the fiber knob of an Ad5 vector (110). The DARPin units block CAR mediated uptake and re-targeted the vector to HER2 or EGFR positive tumor cells in mice (110,111). This combination strategy improved the tumor to liver localization ratio of the Ad5 vector and circumvented intracellular neutralization by TRIM21, thus significantly boosting transgene expression.…”

Designer Oncolytic Adenovirus: Coming of Age

“…Consequently, it is conceivable that ablation of this interaction may result in enhanced clearance of the FX binding ablated virus from the body, reducing therapeutic efficacy [193]. However, mutation of hexon protein to ablate FX interactions could be extended to also inhibit IgM binding, albeit, this may preclude the potentially beneficial effects provided through steric inhibition of hexon interaction by large molecule binding (such as FX), which may represent a broadly effective mechanism for inhibiting immune recognition, as described by Schmid et al (discussed later in this review) [12]. Not all adenoviruses bind FX, highlighting that binteraction with FX is not a universally required mechanism for immune evasion, and this immune evasion could be achieved through incorporation of a hexon or hexon hypervariable regions derived from low seroprevalence adenovirus that are unrecognized by the immune system [194].…”